Antisense modulation of stearoyl-CoA desaturase expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of stearoyl-CoA desaturase. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding stearoyl-CoA desaturase. Methods of using these compounds for modulation of stearoyl-CoA desaturase expression and for treatment of diseases associated with expression of stearoyl-CoA desaturase are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. patentapplication Ser. No. 09/918,187, filed Jul. 30, 2001.

BACKGROUND OF THE INVENTION

Saturated fatty acids are known to be the precursors of unsaturatedfatty acids in higher organisms. However, the control mechanisms thatgovern the conversion of saturated fatty acids to unsaturated fattyacids are not well understood. The relative amounts of different fattyacids have effects on the physical properties of membranes. Furthermore,regulation of unsaturated fatty acids is important because they play arole in cellular activity, metabolism and nuclear events that governgene transcription.

A critical committed step in the biosynthesis of mono-unsaturated fattyacids is the introduction of the first cis-double bond in the delta-9position (between carbons 9 and 10). This oxidative reaction iscatalyzed by stearoyl-CoA desaturase (SCD, also known asdelta-9-desaturase) and involves cytochrome b₅, NADH (P)-cytochrome b₅reductase and molecular oxygen (Ntambi, J. Lipid Res., 1999, 40,1549-1558). Although the insertion of the double bond occurs in severaldifferent methylene-interrupted fatty acyl-CoA substrates, the preferredsubstrates are palmitoyl- and stearoyl-CoA, which are converted topalmitoleoyl- and oleoyl-CoA respectively (Ntambi, J. Lipid Res., 1999,40, 1549-1558).

It has been recognized that, regardless of diet, the major storage fattyacids in human adipose tissue are oleic acid, an 18 carbon unsaturatedfatty acid, and palmitoleic acid, a 16 carbon unsaturated fatty acid(Ntambi, J. Lipid Res., 1999, 40, 1549-1558). During the de novosynthesis of fatty acids, the fatty acid synthase enzyme stops atpalmitoleic acid but the end product of the pathway is usually oleicacid (Ntambi, J. Lipid Res., 1999, 40, 1549-1558).

The stearoyl-CoA desaturase gene was partially characterized in 1994 viaisolation of a 0.76 kb partial cDNA from human adipose tissue (Li etal., Int. J. Cancer, 1994, 57, 348-352). Increased levels ofstearoyl-CoA desaturase mRNA were found in colonic and esophagealcarcinomas and in hepatocellular carcinoma (Li et al., Int. J. Cancer,1994, 57, 348-352). The gene was fully characterized in 1999 and it wasfound that alternative usage of polyadenylation sites generates twotranscripts of 3.9 and 5.2 kb (Zhang et al., Biochem. J., 1999, 340,255-264). Two loci for the stearoyl-CoA desaturase gene were mapped tochromosomes 10 and 17 and it was determined that the chromosome 17 lociencodes a transcriptionally inactive pseudogene (Ntambi, J. Lipid Res.,1999, 40, 1549-1558).

A nucleic acid molecule encoding the human stearoyl-CoA desaturase and anucleic acid molecule, which under suitable conditions, specificallyhybridizes to the nucleic acid molecule encoding the human stearoyl-CoAdesaturase, have been described (Stenn et al., International patentpublication WO 00/09754, 2000).

Stearoyl-CoA desaturase affects the ratio of stearate to oleate, whichin turn, affects cell membrane fluidity. Alterations of this ratio havebeen implicated in various disease states including cardiovasculardisease, obesity, non-insulin-dependent diabetes mellitus, skin disease,hypertension, neurological diseases, immune disorders and cancer(Ntambi, J. Lipid Res., 1999, 40, 1549-1558). Stearoyl-CoA desaturasehas been viewed as a lipogenic enzyme not only for its key role in thebiosynthesis of monounsaturated fatty acids, but also for its pattern ofregulation by diet and insulin (Ntambi, J. Lipid Res., 1999, 40,1549-1558).

The regulation of stearoyl-CoA desaturase is therefore of considerablephysiologic importance and its activity is sensitive to dietary changes,hormonal imbalance, developmental processes, temperature changes,metals, alcohol, peroxisomal proliferators and phenolic compounds(Ntambi, J. Lipid Res., 1999, 40, 1549-1558).

Animal models have been very useful in investigations of the regulationof stearoyl-CoA by polyunsaturated fatty acids (PUFAs). For example, inadipose tissue of lean and obese Zucker rats, a 75% decrease instearoyl-CoA desaturase mRNA was observed when both groups were fed adiet high in PUFAs relative to a control diet (Jones et al., Am. J.Physiol., 1996, 271, E44-49).

Similar results have been obtained with tissue culture systems. In themurine 3T3-L1 adipocyte cell line, arachidonic linoleic, linolenic, andeicosapentanenoic acids decreased stearoyl-CoA desaturase expression ina dose-dependent manner (Sessler et al., J. Biol. Chem., 1996, 271,29854-29858).

The molecular mechanisms by which PUFAs regulate stearoyl-CoA desaturasegene expression in different tissues are still poorly understood. Thecurrent understanding of the regulatory mechanism involves binding ofPUFAs to a putative PUFA-binding protein, after which repressiontranscription occurs via binding of the PUFA-binding protein to acis-acting PUFA response element of the stearoyl-CoA desaturase gene(SREBP) (Ntambi, J. Lipid Res., 1999, 40, 1549-1558; Zhang et al.,Biochem. J., 2001, 357, 183-193).

Cholesterol has also been identified as a regulator of stearoyl-CoAdesaturase gene expression by a mechanism involving repression of thematuration of the sterol regulatory element binding protein (Bene etal., Biochem. Biophys. Res. Commun., 2001, 284, 1194-1198; Ntambi, J.Lipid Res., 1999, 40, 1549-1558).

Thiazolidinediones have been employed as regulators of stearoyl-CoAdesaturase activity in murine 3T3-L1 adipocytes (Kim et al., J. LipidRes., 2000, 41, 1310-1316), and in diabetic rodents (Singh Ahuja et al.,Mol. Pharmacol., 2001, 59, 765-773).

Compositions comprising a saponin in an amount effective to inhibitstearoyl-CoA desaturase enzyme activity were described. The saponin wasderived from a source selected from the group consisting of Quillajasaponaria, Panax trifolium, Panax quinquefolium and Glycyrrhiza glabra(Chavali and Forse, International patent publication No. WO 99/639791999).

An inhibitor of stearoyl-CoA desaturase was prepared in a form suitablefor oral, parenteral, rectal or dermal administration for use inmodifying the lipid structure of cell membranes. The inhibitor wasdescribed as consisting of a saturated fatty acid having from 12 to 28carbon atoms in the alkyl chain, e.g. stearic acid, or apharmaceutically acceptable derivative thereof prepared in a formsuitable for parenteral, rectal or dermal administration (Wood et al.,European Patent No. EP 238198 1987). A stearoyl-CoA desaturase antisensevector has been used to reduce expression levels of stearoyl-CoAdesaturase in chicken LMH hepatoma cells (Diot et al., Arch. Biochem.Biophys., 2000, 380, 243-250).

To date, investigative strategies aimed at inhibiting stearoyl-CoAdesaturase function include the previously cited uses of polyunsaturatedfatty acids, saturated fatty acids, thiazolidinediones, cholesterol, andan antisense vector. However, these strategies are untested astherapeutic protocols. Consequently, there remains a long felt need foradditional agents capable of effectively inhibiting stearoyl-CoAdesaturase.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of stearoyl-CoA desaturase. Such compositions and methodsare shown to modulate the expression of stearoyl-CoA desaturase,including inhibition of both isoforms of stearoyl-CoA desaturase.

In particular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodingstearoyl-CoA desaturase. Such compounds, particularly antisenseoligonucleotides, are targeted to a nucleic acid encoding stearoyl-CoAdesaturase, and modulate the expression of stearoyl-CoA desaturase.Pharmaceutical and other compositions comprising the compounds of theinvention are also provided.

Further provided are methods of modulating the expression ofstearoyl-CoA desaturase in cells or tissues comprising contacting thecells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of stearoyl-CoAdesaturase, by administering a therapeutically or prophylacticallyeffective amount of one or more of the antisense compounds orcompositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding stearoyl-CoA desaturase, ultimatelymodulating the amount of stearoyl-CoA desaturase produced. This isaccomplished by providing antisense compounds that specificallyhybridize with one or more nucleic acids encoding stearoyl-CoAdesaturase.

Antisense technology is emerging as an effective means of reducing theexpression of specific gene products and is uniquely useful in a numberof therapeutic, diagnostic and research applications involvingmodulation of stearoyl-CoA desaturase expression.

As used herein, the terms “target nucleic acid” and “nucleic acidencoding stearoyl-CoA desaturase” encompass DNA encoding stearoyl-CoAdesaturase, RNA (including pre-mRNA and mRNA) transcribed from such DNA,and also cDNA derived from such RNA. The specific hybridization of anoligomeric compound with its target nucleic acid interferes with thenormal function of the nucleic acid. This modulation of function of atarget nucleic acid by compounds that specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression ofstearoyl-CoA desaturase. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In one embodiment of thepresent invention, inhibition is a preferred form of modulation of geneexpression and mRNA is a preferred target.

For example, in one embodiment of the present invention, the compoundsof the present invention inhibit expression of stearoyl-CoA desaturasebyat least 10% as measured in a suitable assay, such as those described inthe examples below. In another embodiment, the compounds of the presentinvention inhibit expression of stearoyl-CoA desaturase by at least 25%.In still another embodiment of the invention, the compounds of thepresent invention inhibit expression of stearoyl-CoA desaturase by atleast 40%. In yet a further embodiment of this invention, the compoundsof the present invention inhibit expression of stearoyl-CoA desaturaseby at least 50%. In a further embodiment of this invention, thecompounds of the present invention inhibit expression of stearoyl-CoAdesaturase by at least 60%. In another embodiment of this invention, thecompounds of the present invention inhibit expression of stearoyl-CoAdesaturase by at least 70%. In still another embodiment of thisinvention, the compounds of this invention inhibit expression ofstearoyl-CoA desaturase by at least 80%. In another embodiment of thisinvention, the compounds of this invention inhibit expression ofstearoyl-CoA desaturase by at least 90% or higher. Exemplary compoundsare illustrated in Examples 15, and 17 to 24 below.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process asdescribed herein begins with the identification of a nucleic acidsequence encoding stearoyl-CoA desaturase. This may be, for example, acellular gene (or mRNA transcribed from the gene). The targeting processalso includes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, results. In oneembodiment of the present invention, a preferred intragenic site is theregion encompassing the translation initiation or termination codon ofthe open reading frame (ORF) of the gene. Since, as is known in the art,the translation initiation codon is typically 5′-AUG (in transcribedmRNA molecules; 5′-ATG in the corresponding DNA molecule), thetranslation initiation codon is also referred to as the “AUG codon,” the“start codon” or the “AUG start codon”. A minority of genes has atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding stearoyl-CoA desaturase, regardless of thesequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is another embodiment of a region ofthe nucleic acid sequence encoding stearoyl-CoA desaturase which may betargeted effectively. Other target regions of this invention include the5′ untranslated region (5′ UTR) of the nucleic acid sequence encodingstearoyl-CoA desaturase, known in the art to refer to the portion of anmRNA in the 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene.Still another target region is the 3′ untranslated region (3′ UTR) ofthe nucleic acid sequence encoding stearoyl-CoA desaturase, known in theart to refer to the portion of an mRNA in the 3′ direction from thetranslation termination codon, and thus including nucleotides betweenthe translation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region of the nucleicacid sequence encoding stearoyl-CoA desaturase may also be a preferredtarget region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, of the nucleic acid sequence encoding stearoyl-CoA desaturasemay also be preferred target regions, and are particularly useful insituations where aberrant splicing is implicated in disease, or where anoverproduction of a particular mRNA splice product is implicated indisease. Aberrant fusion junctions of the nucleic acid sequence encodingstearoyl-CoA desaturase, due to rearrangements or deletions, are alsopreferred targets. In another embodiment of this invention, introns ofthe nucleic acid sequence encoding stearoyl-CoA desaturase can also beeffective target regions for antisense compounds targeted, for example,to DNA or pre-mRNA of the nucleic acid sequence encoding stearoyl-CoAdesaturase.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect. See, e.g., Tables 1-5 below.

For example, Tables 1 and 2 illustrate antisense oligonucleotides thathybridize to target regions of nucleotide 9 to 5100 of the nucleotidesequence of human stearoyl CoA desaturase SEQ ID NO: 3. In oneembodiment, for example, desirable oligonucleotides target regionswithin nucleotides 13 to 71. In another example, desirableoligonucleotides target regions within nucleotides 178 to 247. Inanother example, desirable oligonucleotides target regions withinnucleotides 482 to 843. In another example, desirable oligonucleotidestarget regions within nucleotides 892 to 1064. In another example,desirable oligonucleotides target regions within nucleotides 1303-1502.In another example, desirable oligonucleotides target regions withinnucleotides 1597-2233. In another example, desirable oligonucleotidestarget regions within nucleotides 2245-2589. In another example,desirable oligonucleotides target regions within nucleotides 2676-3278.In another example, desirable oligonucleotides target regions withinnucleotides 3342-3499. In another example, desirable oligonucleotidestarget regions within nucleotides 3655-3674. In another example,desirable oligonucleotides target regions within nucleotides 3707-3790.In another example, desirable oligonucleotides target regions withinnucleotides 3825-3853. In another example, desirable oligonucleotidestarget regions within nucleotides 3911-4072. In another example,desirable oligonucleotides target regions within nucleotides 4132-4224.In another example, desirable oligonucleotides target regions withinnucleotides 4261-4398. In another example, desirable oligonucleotidestarget regions within nucleotides 4420-4554. In another example,desirable oligonucleotides target regions within nucleotides 4645-4677.In another example, desirable oligonucleotides target regions withinnucleotides 4834-4865. In another example, desirable oligonucleotidestarget regions within nucleotides 4892-5100. Oligonucletides that targetany nucleotide sequence within SEQ ID NO: 3, with the explicit exclusionof target regions between nucleotides 70-91, 242-262 and 860-882, areincluded within this invention.

As another example, Table 2 indicates illustrative oligonucleotides thathybridize to target regions found within nucleotides 505 to 14020 of thenucleotide sequence of human stearoyl CoA desaturase SEQ ID NO: 81.

Tables 3-5 illustrate oligonucleotides that bind to target regionswithin nucleotides 1 to 5366 of the nucleotide sequence of mousestearoyl CoA desaturase SEQ ID NO: 222.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases that pairthrough the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the target nucleic acid sequence (DNA or RNA)encoding stearoyl-CoA desaturase.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. An antisense compound is specificallyhybridizable when binding of the compound to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA tocause a loss of utility of the stearoyl-CoA desaturase enzyme. Therealso must be a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-stearoyl-CoAdesaturase target sequences under conditions in which specific bindingis desired. Such conditions include physiological conditions in the caseof in vivo assays or therapeutic treatment, and in the case of in vitroassays, include conditions in which the assays are performed.

For example, in one embodiment, the antisense compounds of the presentinvention comprise at least 80% sequence complementarity to a targetregion within the target nucleic acid of stearoyl-CoA desaturase towhich they are targeted. In another embodiment, the antisense compoundsof the present invention comprise at least 90% sequence complementarityto a target region within the target nucleic acid of stearoyl-CoAdesaturase to which they are targeted. In still another embodiment ofthis invention, the antisense compounds of the present inventioncomprise at least 95% sequence complementarity to a target region withinthe target nucleic acid sequence of stearoyl-CoA desaturase to whichthey are targeted. For example, an antisense compound in which 18 of 20nucleobases of the antisense compound are complementary, and wouldtherefore specifically hybridize, to a target region would represent 90percent complementarity. Percent complementarity of an antisensecompound with a region of a target nucleic acid can be determinedroutinely using basic local alignment search tools (BLAST programs)(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Antisense and other compounds of the invention that hybridize to thetarget and inhibit expression of stearoyl-CoA desaturase are identifiedas taught herein. In one embodiment of this invention, the sequences ofthese compounds are hereinbelow identified as preferred embodiments ofthe invention (see, e.g., Tables 1-5). The target sites to which thesepreferred sequences are complementary are hereinbelow referred to as“active sites” and are therefore preferred sites for targeting (see,e.g., Tables 1-5). Therefore another embodiment of the inventionencompasses compounds that hybridize to these active sites.

In one embodiment of this invention, the term “illustrative targetregion” is defined as a nucleobase sequence of a target region ofstearoyl-CoA desaturase, to which an active antisense compound istargeted. For example, an illustrative target region may be at least 8or at least 15 nucleobases in length. In still another embodiment anillustrative target region is at least 25 nucleobases of the nucleicacid sequence or molecule encoding stearoyl-CoA desaturase, to which anactive antisense compound is targeted. In still another embodiment anillustrative target region is at 35 nucleobases. In yet anotherembodiment an illustrative target region is at least 50 nucleobases ofthe nucleic acid sequence or molecule encoding stearoyl-CoA desaturase,to which an active antisense compound is targeted. In still anotherembodiment an illustrative target region is at least 70 nucleobases. Inanother embodiment an illustrative target region is at least 80nucleobases or more. In still another embodiments, the illustrativetarget regions consist of consecutive nucleobases of the lengthsidentified above.

Exemplary additional target regions include DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 5′-terminus ofone of the illustrative target regions (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target region and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlyadditional target regions are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative target regions (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the target region and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

For use in kits and diagnostics, the antisense compounds of the presentinvention, either alone or in combination with other antisense compoundsor therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

Expression patterns within cells or tissues treated with one or moreantisense compounds are compared to control cells or tissues not treatedwith antisense compounds and the patterns produced are analyzed fordifferential levels of gene expression as they pertain, for example, todisease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds that affect expressionpatterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals, particularly mammals, and including man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides that hybridize to thetarget nucleic acid encoding stearoyl-CoA desaturase and modulateexpression of that enzyme.

The antisense compounds in accordance with this invention preferablycomprise from at least 8 nucleobases (i.e. about 8 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides. In one embodiment, antisense compounds of thisinvention are antisense oligonucleotides of at least about 15nucleobases in length. In another embodiment, antisense compounds ofthis invention comprise about 25 nucleobases in length. In still anotherembodiment, antisense compounds of this invention comprise about 35nucleobases in length. In yet another embodiment, antisense compounds ofthis invention comprise about 40 nucleobases in length. In still anotherembodiment, antisense compounds of this invention comprise about 50nucleobases in length. In another embodiment, antisense compounds ofthis invention comprise about 60 nucleobases in length. In still anotherembodiment, antisense compounds of this invention comprise about 70nucleobases in length. In yet another embodiment, antisense compounds ofthis invention comprise about 80 nucleobases in length.

In other embodiments, exemplary antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative antisense compounds (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the antisensecompound which is specifically hybridizable to the target nucleic acidand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly, in another embodiment, such antisense compoundsinclude at least 12 consecutive nucleobases from the 5′-terminus of oneof the illustrative antisense compounds. In yet another embodiment, theantisense compound includes at least 25 consecutive nucleobases from the5′-terminus of one of the illustrative antisense compounds. In a furtherembodiment, the antisense compound includes at least 30 consecutivenucleobases from the 5′-terminus of one of the illustrative antisensecompounds. In yet another embodiment, the antisense compound includes atleast 50 consecutive nucleobases from the 5′-terminus of one of theillustrative antisense compounds. In still another embodiment, theantisense compound includes at least 60 or more consecutive nucleobasesfrom the 5′-terminus of one of the illustrative antisense compounds.

Similarly in another embodiment antisense compounds are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds (the remaining nucleobases being a consecutive stretch of thesame DNA or RNA beginning immediately downstream of the 3′-terminus ofthe antisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). In another embodiment, such antisense compoundsinclude at least 12 consecutive nucleobases from the 3′-terminus of oneof the illustrative antisense compounds. In yet another embodiment, theantisense compound includes at least 25 consecutive nucleobases from the3′-terminus of one of the illustrative antisense compounds. In a furtherembodiment, the antisense compound includes at least 30 consecutivenucleobases from the 3′-terminus of one of the illustrative antisensecompounds. In yet another embodiment, the antisense compound includes atleast 50 consecutive nucleobases from the 3′-terminus of one of theillustrative antisense compounds. In still another embodiment, theantisense compound includes at least 60 or more consecutive nucleobasesfrom the 3′-terminus of one of the illustrative antisense compounds. Onehaving skill in the art, once armed with the antisense compoundsillustrated, and other teachings herein will be able, without undueexperimentation, to identify further antisense compounds of thisinvention.

Specific sequences of particular exemplary target regions ofstearoyl-CoA desaturase and representative antisense and other compoundsof the invention, which hybridize to the target, and inhibit expressionof the target, are identified below are set forth below in Tables 1-5.One of skill in the art will recognize that these serve to illustrateand describe particular embodiments within the scope of the presentinvention. Once armed with the teachings of the illustrative targetregions described herein may without undue experimentation identifyfurther target regions, as described above. In addition, one havingordinary skill in the art using the teachings contained herein will alsobe able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these illustrativetarget regions using techniques available to the ordinary practitionerin the art.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and borano-phosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage, i.e. a single inverted nucleosideresidue that may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in International patent publication Nos. WO 98/39352 and WO99/14226.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. The compounds of the invention caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Publication No. WO93/07883 (ApplicationPCT/US92/09196, filed Oct. 23, 1992) the entire disclosure of which isincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999), which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in International Patent Publication Nos. WO 93/24510 toGosselin et al., published Dec. 9, 1993 or WO 94/26764, and U.S. Pat.No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of the acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder that can be treated by modulating theexpression of stearoyl-CoA desaturase is treated by administeringantisense compounds in accordance with this invention. Among suchdiseases or disorder are included, for example, cardiovascular disease,obesity, non-insulin-dependent diabetes mellitus, skin disease,hypertension, neurological diseases, immune disorders and cancer.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically to prevent such diseases or disorders, e.g., to preventor delay infection, undue weight gain, inflammation or tumor formation,for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingstearoyl-CoA desaturase, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encodingstearoyl-CoA desaturase can be detected by means known in the art. Suchmeans may include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofstearoyl-CoA desaturase in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters include but are not limitedarachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. patent applicationSer. No. 09/315,298 filed on May 20, 1999, which is incorporated hereinby reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers, surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. Published patent application No. 2003/0040497 (Feb. 27, 2003) andits parent applications; U.S. Published patent application No.2003/0027780 (Feb. 6, 2003) and its parent applications; and U.S. patentapplication Ser. No. 09/082,624 (filed May 21, 1998), each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutions,which may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug, which may be present as a solution in either theaqueous phase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, cited above).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, cited above). Surfactants aretypically amphiphilic and comprise a hydrophilic and a hydrophobicportion. The ratio of the hydrophilic to the hydrophobic nature of thesurfactant has been termed the hydrophile/lipophile balance (HLB) and isa valuable tool in categorizing and selecting surfactants in thepreparation of formulations. Surfactants may be classified intodifferent classes based on the nature of the hydrophilic group:nonionic, anionic, cationic and amphoteric (Rieger, cited above).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, cited above).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture has been reviewed inthe literature (Idson, cited above). Emulsion formulations for oraldelivery have been very widely used because of reasons of ease offormulation, efficacy from an absorption and bioavailability standpoint(Rosoff, cited above; Idson, cited above). Mineral-oil base laxatives,oil-soluble vitamins and high fat nutritive preparations are among thematerials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphile,which is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, cited above). Typically microemulsions aresystems that are prepared by first dispersing an oil in an aqueoussurfactant solution and then adding a sufficient amount of a fourthcomponent, generally an intermediate chain-length alcohol to form atransparent system. Therefore, microemulsions have also been describedas thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, citedabove; Block, cited above). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., 1994 cited above; Ho et al., J. Pharm. Sci., 1996, 85, 138-143).Often microemulsions may form spontaneously when their components arebrought together at ambient temperature. This may be particularlyadvantageous when formulating thermolabile drugs, peptides oroligonucleotides. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome that is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, cited above). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes that interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes that are pH-sensitive or negatively-charged, entrap DNA ratherthan complex with it. Since both the DNA and the lipid are similarlycharged, repulsion rather than complex formation occurs. Nevertheless,some DNA is entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes. This latterterm, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 andInternational Patent Publication No. WO 88/04924, both to Allen et al.,disclose liposomes comprising (1) sphingomyelin and (2) the gangliosideG_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152(Webb et al.) discloses liposomes comprising sphingomyelin. Liposomescomprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed inInternational Patent Publication No. WO 97/13499 (Lim et al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and International Patent Publication No. WO 90/04384 to Fisher. Liposomecompositions containing 1-20 mole percent of PE derivatized with PEG,and methods of use thereof, are described by Woodle et al. (U.S. Pat.Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804and European Patent No. EP 0 496 813 B1). Liposomes comprising a numberof other lipid-polymer conjugates are disclosed in International PatentPublication No. WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in International Patent Publication No. WO 94/20073(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids aredescribed in International Patent Publication No. WO 96/10391 (Choi etal.) . U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No.5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can befurther derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. International Patent Publication No. WO 96/40062 to Thierry et al.discloses methods for encapsulating high molecular weight nucleic acidsin liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. disclosesprotein-bonded liposomes and asserts that the contents of such liposomesmay include an antisense RNA. U.S. Patent No. 5,665,710 to Rahman et al.describes certain methods of encapsulating oligodeoxynucleotides inliposomes. International Patent Publication No. WO 97/04787 to Love etal. discloses liposomes comprising antisense oligonucleotides targetedto the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid droplets thatare so highly deformable that they are easily able to penetrate throughpores that are smaller than the droplet. Transfersomes are adaptable tothe environment in which they are used, e.g. they are self-optimizing(adaptive to the shape of pores in the skin), self-repairing, frequentlyreach their targets without fragmenting, and often self-loading. To maketransfersomes it is possible to add surface edge-activators, usuallysurfactants, to a standard liposomal composition. Transfersomes havebeen used to deliver serum albumin to the skin. Thetransfersome-mediated delivery of serum albumin has been shown to be aseffective as subcutaneous injection of a solution containing serumalbumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger,cited above).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, cited above).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., 1991, cited above); and perfluorochemical emulsions, such asFC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., ,1991, p.92, cited above; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., 1991, page 92, cited above; Swinyard, Chapter 39 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J.Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci.,1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediamine tetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., 1991, page 92, cited above; Muranishi, 1990, cited above; Buuret al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, 1990, citedabove). This class of penetration enhancers include, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacycloalkanonederivatives (Lee et al., 1991, page 92, cited above); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al.,International Patent Publication No. WO 97/30731), are also known toenhance the cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration that do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration that do not deleteriously react with nucleic acids can beused.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 g to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Mass. or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.)

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl)nucleoside amidites and2′-O-(dimethylaminooxyethyl)nucleoside amidites2′-(Dimethylaminooxyethoxy)nucleoside amidites

2′-(Dimethylaminooxyethoxy)nucleoside amidites [also known in the art as2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl)thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was stirred for 1 h. Solvent was removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyll-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N₁,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy)nucleoside amidites

2′-(Aminooxyethoxy)nucleoside amidites [also known in the art as2′-O-(aminooxyethyl)nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl)diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee et al., International PatentPublication No. WO 94/02501). Standard protection procedures shouldafford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2 (2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate.

Evaporation of the solvent followed by silica gel chromatography usingMeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine) gives the titlecompound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P=O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished International Patent Publication Nos. WO 94/17093 and WO94/02499, herein incorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3 Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

Peptide nucleic acids (PNAS) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]ChimericOligonucleotides

(2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85%full-length material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing seven cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

T-24 cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 μg/mL (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 μg/mL (Gibco/Life Technologies, Gaithersburg, Md.).Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal calf serum,non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

AML12 Cells:

AML12 (alpha mouse liver 12) cell line was established from hepatocytesfrom a mouse (CD1 strain, line MT42) transgenic for human TGF alpha.Cells are cultured in a 1:1 mixture of Dulbecco's modified Eagle'smedium and Ham's F12 medium with 0.005 mg/ml insulin, 0.005 mg/mltransferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone, and 90%; 10%fetal bovine serum. For subculturing, spent medium is removed; and freshmedia of 0.25% trypsin, 0.03% EDTA solution is added. Fresh trypsinsolution (1 to 2 ml) is added and the culture is left to sit at roomtemperature until the cells detach.

Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #3872) at a density of 7000 cells/well for use inRT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

Primary Mouse Hepatocytes:

Primary mouse hepatocytes were prepared from CD-1 mice purchased fromCharles River Labs (Wilmington, Mass.) and were routinely cultured inHepatoyte Attachment Media (Gibco) supplemented with 10% Fetal BovineSerum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone(Sigma), and 10 nM bovine insulin (Sigma). Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analyses, cells are plated onto 100 mm orother standard tissue culture plates coated with rat tail collagen (200μg/mL) (Becton Dickinson) and treated similarly using appropriatevolumes of medium and oligonucleotide.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 medium containing 3.75 μg/mLLIPOFECTIN™ reagent (Gibco BRL) and the desired concentration ofoligonucleotide. After 4-7 hours of treatment, the medium was replacedwith fresh medium. Cells were harvested 16-24 hours afteroligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of Oligonucleotide Inhibition of Stearoyl-CoADesaturase Expression

Antisense modulation of stearoyl-CoA desaturase expression can beassayed in a variety of ways known in the art. For example, stearoyl-CoAdesaturase mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are taught in, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysisis routine in the art and is taught in, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9,John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

Protein levels of stearoyl-CoA desaturase can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to stearoyl-CoA desaturase canbe identified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional antibody generation methods. Methods forpreparation of polyclonal antisera are taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11 —Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS, 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of stearoyl-CoADesaturase mRNA Levels

Quantitation of stearoyl-CoA desaturase mRNA levels was determined byreal-time quantitative PCR using the ABI PRISM™ 7700 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., JOE, FAM, or VIC, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda,Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISM™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1× TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™ reagent, and12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25μL total RNA solution. The RT reaction was carried out by incubation for30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the AMPLITAQ GOLD™ reagent, 40 cycles of a two-step PCRprotocol were carried out: 95° C. for 15 seconds (denaturation) followedby 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ reagent(Molecular Probes, Inc. Eugene, OR). GAPDH expression is quantified byreal time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ reagent are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 25 uL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nmand emission at 520 nm.

Probes and primers to human stearoyl-CoA desaturase were designed tohybridize to a human stearoyl-CoA desaturase sequence, using publishedsequence information (GenBank accession number AF097514, incorporatedherein as SEQ ID NO:3). For human stearoyl-CoA desaturase the PCRprimers were: forward primer: GATCCCGGCATCCGAGA (SEQ ID NO: 4) reverseprimer: GGTATAGGAGCTAGAGATATCGTCCTG (SEQ ID NO: 5) and the PCR probewas: FAM-CCAAGATGCCGGCCCACTTGC-TAMRA (SEQ ID NO: 6) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For human GAPDH the PCR primers were:

-   forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where    JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent    reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.)    is the quencher dye.

Example 14 Northern Blot Analysis of Stearoyl-CoA Desaturase mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ reagent (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 apparatus (Stratagene, Inc, La Jolla,Calif.) and then robed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions.

To detect human stearoyl-CoA desaturase, a human stearoyl-CoA desaturasespecific probe was prepared by PCR using the forward primerGATCCCGGCATCCGAGA (SEQ ID NO: 4) and the reverse primerGGTATAGGAGCTAGAGATATCGTCCTG (SEQ ID NO:5). To normalize for variationsin loading and transfer efficiency membranes were stripped and probedfor human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA(Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ apparatus and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

EXAMPLE 15 Antisense Inhibition of Human Stearoyl-CoA DesaturaseExpression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswas designed to target different regions of the human stearoyl-CoAdesaturase RNA, using published sequence (GenBank accession numberAF097514, incorporated herein as SEQ ID NO: 3). The oligonucleotides areshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the oligonucleotidebinds. All compounds in Table 1 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Thecompounds were analyzed for their effect on human stearoyl-CoAdesaturase mRNA levels in HepG2 cells by quantitative real-time PCR asdescribed in other example herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”. TABLE 1 Inhibitionof human stearoyl-CoA desaturase mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO147899 5′UTR 3 9 GTCCGGTATTTCCTCAGCCC N.D. 10 147900 5′UTR 3 72CCGCGGTGCGTGGAGGTCCC N.D. 11 147901 5′UTR 3 121 TACGCGCTGAGCCGCGGCGCN.D. 12 147902 5′UTR 3 141 GCGGTTTCGAAGCCCGCCGG N.D. 13 147903 Coding 3311 CCTCCATTCTGCAGGACCCT N.D. 14 147904 Coding 3 471TCCCAAGTGTAGCAGAGACA N.D. 15 147905 Coding 3 571 CTCCTGCTGTTATGCCCAGGN.D. 16 147906 Coding 3 691 CACGGTGGTCACGAGCCCAT N.D. 17 147907 Coding 3771 CAGCCAACCCACGTGAGAGA 22 18 147908 Coding 3 824 GACAAGTCTAGCGTACTCCC49 19 147909 Coding 3 1011 GTTCACCAGCCAGGTGGCAT 10 20 147910 Coding 31111 TGTGGAAGCCCTCACCCACA 0 21 147911 Coding 3 1171 AGTTGATGTGCCAGCGGTAC6 22 147912 Stop 3 1307 GGACCCCAAACTCAGCCACT 25 23 Codon 147913 3′UTR 31581 TGCCTGGGAGGCAATAAGGG 8 24 147914 3′UTR 3 1861 ATACATGCTAACTCTCTCCC10 25 147915 3′UTR 3 1941 AAGTCCTCATTAGGTAGGCA 37 26 147916 3′UTR 3 2241TGTAATGAGCAGCTCATGGA 0 27 147917 3′UTR 3 2616 TCAGTAACCTTCTCAAGCCC 0 28147918 3′UTR 3 2980 GGAGCTGCCTGGACAGCAAG 16 29 147919 3′UTR 3 3011TCAGTGACCCTGAGCATTCT 30 30 147920 3′UTR 3 3231 TGGCTGGCCCACTGGCTCAA 1131 147921 3′UTR 3 3291 GCATGCCCTCTGGTTCTGAC 7 32 147922 3′UTR 3 3471GCTTTGCAGTTCACCCTGAC 23 33 147923 3′UTR 3 3502 GTGGTATCTCAAATCCCAGG 0 34147924 3′UTR 3 3791 TAGTCCAGGCTAACCCCTGT 0 35 147925 3′UTR 3 3851GTGATCTTCCCTTAGATCCT 0 36 147926 3′UTR 3 4101 CTCAGCAGACACACTCCCAC 3 37147927 3′UTR 3 4226 GCTAAGTTGTCAGCACACCC 0 38 147928 3′UTR 3 4406AAGTTTCCAGAATGAAGCCC 25 39 147929 3′UTR 3 4571 AGAGAATACACCCAAGATAC 0 40147930 3′UTR 3 4708 TAGTTAAGTGACTTGCCCAG 0 41 147931 3′UTR 3 4771GCCCTTTGAGGTAGGTCAGT 4 42 147932 3′UTR 3 4921 CCATATAGACTAATGACAGC 10 43147933 3′UTR 3 5021 CTGTATGTTTCCGTGGCAAT 11 44 168231 5′UTR 3 101CTTGCACGCTAGCTGGTTGT N.D. 45 168232 Coding 3 331 GCATCGTCTCCAACTTATCTN.D. 46 168233 Coding 3 451 TAAGGATGATGTTTCTCCAG N.D. 47 168234 Coding 3526 CCCAAAGCCAGGTGTAGAAC N.D. 48 168235 Coding 3 601TGTAAGAGCGGTGGCTCCAC N.D. 49 168236 Coding 3 661 CATTCTGGAATGCCATTGTGN.D. 50 168237 Coding 3 731 TTATGAGGATCAGCATGTGT N.D. 51 168238 Coding 3861 CCTCTGGAACATCACCAGTT N.D. 52 168239 Coding 3 901GGATGAAGCACATCAGCAGC N.D. 53 168240 Coding 3 936 TTCACCCCAGAAATACCAGGN.D. 54 168241 Coding 3 1082 GAAACCAGGATATTCTCCCG N.D. 55 168242 Coding3 1151 TCACTGGCAGAGTAGTCATA N.D. 56 168243 Coding 3 1261TAATCCTGGCCAAGATGGCG N.D. 57 168244 3′UTR 3 1401 TCATCATCTTTAGCATCCTGN.D. 58 168245 3′UTR 3 1601 GCAAAGACTGACCAGCTGCT N.D. 59 168246 3′UTR 31748 GACTACCCAGAAGATTCTGT N.D. 60 168247 3′UTR 3 1881CTTCCCTCATCCTTACATTC N.D. 61 168248 3′UTR 3 1985 CCCGAGCCAGGAGAGAAAGGN.D. 62 168249 3′UTR 3 2102 CTTCCCCAGCAGAGACCACT N.D. 63 168250 3′UTR 32281 CCAATATCCTGAAGATGGCA N.D. 64 168251 3′UTR 3 2481CCCAACTAATTCCTCCTCTC N.D. 65 168252 3′UTR 3 2541 TATAGATCCTGTCCCTCAGCN.D. 66 168253 3′UTR 3 2631 CTCCCAATAACTCACTCAGT N.D. 67 168254 3′UTR 32826 AAGAGATTCCTAACCCTGCC N.D. 68 168255 3′UTR 3 2941CACACAAAGGAGGCTGCCTG N.D. 69 168256 3′UTR 3 3051 AAGTGGCAGCTAGCTCTACTN.D. 70 168257 3′UTR 3 3321 CACCCTCACCAAGTAAGCAG N.D. 71 168258 3′UTR 33401 TGCTTCTTCCCAGTGAGAAC N.D. 72 168259 3′UTR 3 3941ATCAAGCAGGCATCTGATGA N.D. 73 168260 3′UTR 3 4052 CCCTCAGCCTGAGGTGCCATN.D. 74 168261 3′UTR 3 4357 ATAATCCTCCACTCAGGCCC N.D. 75 168262 3′UTR 34431 CACTTAAGAAAAGCAGCCCT N.D. 76 168263 3′UTR 3 4681CAGCAAGTCAGTGGCACAGT N.D. 77 168264 3′UTR 3 4971 GGCTAGTTATCCACCGCTTCN.D. 78 168265 3′UTR 3 5044 CCCAATCACAGAAAAGGCAT N.D. 79 168266 3′UTR 35081 AACTACTATATCCCACATAA N.D. 80

As shown in Table 1, SEQ ID NOs 18, 19, 20, 23, 25, 26, 29, 30, 31, 33,39, 43 and 44 demonstrated at least 10% inhibition of human stearoyl-CoAdesaturase expression in this assay. The target sites to which thesepreferred sequences are complementary are herein referred to as “activesites” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 16 Western Blot Analysis of Stearoyl-CoA Desaturase ProteinLevels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to stearoyl-CoAdesaturase is used, with a radiolabelled or fluorescently labeledsecondary antibody directed against the primary antibody species. Bandsare visualized using a PHOSPHORIMAGER™ apparatus(Molecular Dynamics,Sunnyvale Calif.).

Example 17 Antisense Inhibition of Human Stearoyl-CoA DesaturaseExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswas designed to target different regions of the human stearoyl-CoAdesaturase RNA, using published sequence (GenBank accession numberAF097514, incorporated herein as SEQ ID NO: 3 and nucleotides 7371062 to7389569 of the nucleotide sequence with the GenBank accession numberNT_(—)030059.7, incorporated herein as SEQ ID NO: 81). Theoligonucleotides are shown in Table 2. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanstearoyl-CoA desaturase mRNA levels in HepG2 cells by quantitativereal-time PCR as described in other examples herein. The positivecontrol oligonucleotide is ISIS 18078 (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:82), a 2′-O-methoxyl gapmer (2′-O-methoxyethyls shown in bold) with arothioate backbone, which is targeted to human Jun-N-terminal kinase-2(JNK2). Data are averages from two experiments and are shown in Table 2.If present, “N.D.” indicates “no data”. TABLE 2 Inhibition of humanstearoyl-CoA desaturase mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap Seq Control ISISTarget Target % ID SEQ ID # Region Seq ID Site SEQUENCE Inhib No NO300870 5′UTR 3 13 ccgtgtccggtatttcctca 53 83 82 300871 5′UTR 3 25ggcaacgggtgaccgtgtcc 75 84 82 300872 5′UTR 3 42 atttaaaggctagagctggc 6085 82 300873 5′UTR 3 52 cgagccgggaatttaaaggc 40 86 82 300874 5′UTR 3 178gaggctccggagcggagttc 63 87 82 300875 5′UTR 3 215 ttggctctcggatgccggga 6988 82 300876 Start Codon 3 228 gtgggccggcatcttggctc 54 89 82 300877Coding 3 239 tcctgcagcaagtgggccgg 88 90 82 300878 Coding 3 253agctagagatatcgtcctgc 82 91 82 300879 Coding 3 482 ccatacagggctcccaagtg42 92 82 300880 Coding 3 513 gtagaacttgcaggtaggaa 57 93 82 300881 Coding3 566 gctgttatgcccagggcact 93 94 82 300882 Coding 3 667agacatcattctggaatgcc 76 95 82 300883 Coding 3 709 ctgaaaacttgtggtgggca42 96 82 300884 Coding 3 715 gtgtttctgaaaacttgtgg 60 97 82 300885 Coding3 821 aagtctagcgtactcccctt 69 98 82 300886 Coding 3 873tttgtagtacctcctctgga 36 99 82 300887 Coding 3 1045 cataaggacgatatccgaag52 100 82 300888 Stop Codon 3 1303 cccaaactcagccactcttg 50 101 82 3008893′UTR 3 1347 aaacctctgcctggctggtt 87 102 82 300890 3′UTR 3 1381gtagcattattcagtagtta 58 103 82 300891 3′UTR 3 1419 tactggaatgggttaacatc42 104 82 300892 3′UTR 3 1484 tcagcttagcatcataaagg 71 105 82 3008933′UTR 3 1597 agactgaccagctgcttgcc 45 106 82 300894 3′UTR 3 1613gctggacactgagcaaagac 65 107 82 300895 3′UTR 3 1620 tttggaagctggacactgag51 108 82 300896 3′UTR 3 1668 tctggagcaaagaccattcg 91 109 82 3008973′UTR 3 1704 cttcaaagctcacaacagct 69 110 82 300898 3′UTR 3 1711ccacctacttcaaagctcac 68 111 82 300899 3′UTR 3 1716 tcaagccacctacttcaaag45 112 82 300900 3′UTR 3 1723 ctctagctcaagccacctac 57 113 82 3009013′UTR 3 1814 tgtgtcaaatgaagttgctt 66 114 82 300902 3′UTR 3 1842cccgacaatttacctgcttt 75 115 82 300903 3′UTR 3 1869 ttacattcatacatgctaac22 116 82 300904 3′UTR 3 1915 tgtctgatcatggcgagagg 90 117 82 3009053′UTR 3 1969 aaggaagcatgctatgtggt 87 118 82 300906 3′UTR 3 1976ggagagaaaggaagcatgct 81 119 82 300907 3′UTR 3 2040 aactatatgttgcggcattg59 120 82 300908 3′UTR 3 2781 tagatgttaacagagacccc 15 121 82 3009093′UTR 3 2839 aatcagggtagtgaagagat 27 122 82 300910 3′UTR 3 2859gggtagagccaggaatcaag 32 123 82 300911 3′UTR 3 3020 agcagtggttcagtgaccct83 124 82 300912 3′UTR 3 3035 tactttcaaaagagaagcag 27 125 82 3009133′UTR 3 3056 cgtgaaagtggcagctagct 81 126 82 300914 3′UTR 3 3122ccttgtcttgagccatcagt 77 127 82 300915 3′UTR 3 3132 ggtttgccagccttgtcttg78 128 82 300916 3′UTR 3 3222 cactggctcaacatgagcgc 71 129 82 3009173′UTR 3 3238 tgctctgtggctggcccact 93 130 82 300918 3′UTR 3 3252aataaaccctcttttgctct 52 131 82 300919 3′UTR 3 3259 gactgaaaataaaccctctt54 132 82 300920 3′UTR 3 3342 agagcactgactcaggcggg 81 133 82 3009213′UTR 3 3357 ttgcactgccagctgagagc 88 134 82 300922 3′UTR 3 3371tacttctacaagcattgcac 83 135 82 300923 3′UTR 3 3383 actgtttcctcctacttcta63 136 82 300924 3′UTR 3 3409 cttgcccttgcttcttccca 70 137 82 3009253′UTR 3 3432 tttcgaggtgaggcacttgg 60 138 82 300926 3′UTR 3 3480tcagccaaagctttgcagtt 77 139 82 300927 3′UTR 3 3655 ttctgctttgatgactgagc86 140 82 300928 3′UTR 3 2052 atcctcggcctcaactatat 58 141 82 3009293′UTR 3 2136 tccttgttattaaagaaaaa 35 142 82 300930 3′UTR 3 2146ctaagaaatctccttgttat 28 143 82 300931 3′UTR 3 2162 cttcttgatatatgaactaa11 144 82 300932 3′UTR 3 2171 acttcaagacttcttgatat 45 145 82 3009333′UTR 3 2214 aaattccatgagctgctgtt 27 146 82 300934 3′UTR 3 2245gaactgtaatgagcagctca 36 147 82 300935 3′UTR 3 2272 tgaagatggcagagcagaaa27 148 82 300936 3′UTR 3 2321 tggaaatgccacagccatct 70 149 82 3009373′UTR 3 2361 cgacttcacctccttaaatc 56 150 82 300938 3′UTR 3 2397gcaatgtatatatgtatata 31 151 82 300939 3′UTR 3 2506 ccagcagtggagaggaaatt27 152 82 300940 3′UTR 3 2525 cagcctctccatctcatgtc 61 153 82 3009413′UTR 3 2570 ctatgtgaagttcgctctta 66 154 82 300942 3′UTR 3 2589cgtgttctcagatcccttcc 0 155 82 300943 3′UTR 3 2676 aactaattaatgaatggacc36 156 82 300944 3′UTR 3 2700 ttactcatttcaaggagaaa 54 157 82 3009453′UTR 3 2715 gaagccttctagtttttact 55 158 82 300946 3′UTR 3 2726cactgtggagagaagccttc 71 159 82 300947 3′UTR 3 2732 gcacaacactgtggagagaa58 160 82 300948 3′UTR 3 3679 aatcttaatagagcaaagcc 0 161 82 300949 3′UTR3 3707 gactgagtgtttggtagtgt 26 162 82 300950 3′UTR 3 3771agcctctacgcaattaacac 38 163 82 300951 3′UTR 3 3825 ctgaggtgaatagctcaaaa51 164 82 300952 3′UTR 3 3834 ccttttctactgaggtgaat 65 165 82 3009533′UTR 3 3911 tagaaataccagcagacatt 37 166 82 300954 3′UTR 3 3993gcacacgattacaataggaa 62 167 82 300955 3′UTR 3 3999 tccatggcacacgattacaa64 168 82 300956 3′UTR 3 4004 tcagatccatggcacacgat 54 169 82 3009573′UTR 3 4041 aggtgccatccagccttatg 12 170 82 300958 3′UTR 3 4053gccctcagcctgaggtgcca 21 171 82 300959 3′UTR 3 4132 agctttagaatcttgaaaat25 172 82 300960 3′UTR 3 4150 aatgtgtcacttgaattgag 26 173 82 3009613′UTR 3 4193 ctgttagaaatccggactct 33 174 82 300962 3′UTR 3 4205ccaaagcagggactgttaga 41 175 82 300963 3′UTR 3 4261 caacactgtgattagaaaag20 176 82 300964 3′UTR 3 4321 cttcagtagggtctcaggtg 43 177 82 3009653′UTR 3 4331 ctaccagccacttcagtagg 37 178 82 300966 3′UTR 3 4347actcaggcccctttttctac 34 179 82 300967 3′UTR 3 4364 gatactgataatcctccact18 180 82 300968 3′UTR 3 4379 aatcctgcaaatcgtgatac 34 181 82 3009693′UTR 3 4420 agcagccctaacaaaagttt 34 182 82 300970 3′UTR 3 4535aaattttccattttaaatgc 23 183 82 300971 3′UTR 3 4578 cacttacagagaatacaccc38 184 82 300972 3′UTR 3 4584 gagctacacttacagagaat 26 185 82 3009733′UTR 3 4628 aacatggccacctcgctttt 16 186 82 300974 3′UTR 3 4645gccttaaccaccagcataac 40 187 82 300975 3′UTR 3 4653 aggccctggccttaaccacc60 188 82 300976 3′UTR 3 4658 tggagaggccctggccttaa 55 189 82 3009773′UTR 3 4786 tcatgcctcaaaactgccct 34 190 82 300978 3′UTR 3 4800ctaaaaagcattagtcatgc 0 191 82 300979 3′UTR 3 4834 agaattcctgtgctgaagga13 192 82 300980 3′UTR 3 4841 ggtcttgagaattcctgtgc 47 193 82 3009813′UTR 3 4846 actcaggtcttgagaattcc 43 194 82 300982 3′UTR 3 4868ggacattcctattataaaaa 9 195 82 300983 3′UTR 3 4892 acacggacgtatcaagttca41 196 82 300984 3′UTR 3 5024 cctctgtatgtttccgtggc 67 197 82 300985 exon81 505 tgcgaggagttgactggcgc 39 198 82 300986 exon 81 512ggcaaagtgcgaggagttga 45 199 82 300987 exon: intron 81 799ggaaactcacatcgtcctgc 33 200 82 300988 exon: intron 81 1614ggctgcttaccccaaagcca 29 201 82 300989 intron 81 2854ctcagttgcatttcactgta 45 202 82 300990 intron 81 3557gtgggaagagaagatgtcca 4 203 82 300991 intron 81 5287 gccttctctaaggttttaag50 204 82 300992 intron: exon 81 5633 tagaatacccctgccaggag 34 205 82300993 exon: intron 81 5764 aacttcttacctggaatgcc 18 206 82 300994 intron81 7232 ccttgcaaaagagctcatac 56 207 82 300995 exon: intron 81 7900cttcactcacctcctctgga 0 208 82 300996 intron 81 8630 tttgcactgtctctccccac13 209 82 300997 intron 81 8878 tcagtggtttcttacacttg 77 210 82 300998intron: exon 81 9799 tttgtagtacctacattgac 4 211 82 300999 exon: intron81 10032 gctgacttacccacagctcc 10 212 82 301000 intron 81 10163tactgccccctaattttata 0 213 82 301001 intron 81 12377ccatttgcgatacaggaaac 27 214 82 301002 intron: exon 81 14001aagccctcacctgaaacaaa 22 215 82

As shown in Table 2, SEQ ID NOs 83, 84, 85, 87, 88, 89, 90, 91, 93, 94,95, 97, 98, 100, 101, 102, 103, 105, 107, 108, 109, 110, 111, 113, 114,115, 117, 118, 119, 120, 124, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 149, 150, 153, 154, 157, 158,159, 160, 164, 165, 167, 168, 169, 188, 189, 197, 204, 207 and 210demonstrated at least 50% inhibition of human stearoyl-CoA desaturaseexpression in this assay. Preferred antisense oligonucleotide sequencesare SEQ ID NOs 94, 130, 140 and 134. The target sites to which thesepreferred sequences are complementary are herein referred to as “activesites” and are therefore preferred sites for targeting by compounds ofthe present invention.

Example 18 Antisense Inhibition of Mouse Stearoyl-CoA DesaturaseExpression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswas designed to target different regions of the mouse stearoyl-CoAdesaturase RNA, using published sequence (GenBank Accession numberM21280.1, incorporated herein as SEQ ID NO: 216; GenBank Accessionnumber M21281.1, incorporated herein as SEQ ID NO: 217; GenBankAccession number M21282.1, incorporated herein as SEQ ID NO: 218;GenBank Accession number M21283.1, incorporated herein as SEQ ID NO:219; GenBank Accession number M21284.1, incorporated herein as SEQ IDNO: 220; GenBank Accession number M21285.1, incorporated herein as SEQID NO: 221; the concatenation of SEQ ID NOs 216, 217, 218, 219, 220 and221, incorporated herein as SEQ ID NO: 222). The oligonucleotides areshown in Table 3. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target sequence to which the oligonucleotidebinds. All compounds in Table 3 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. TABLE 3Chimeric phosphorothioate oligonucleotides hav- ing 2′-MOE wings and adeoxy gap targeted to mouse stearoyl-CoA Target SEQ ISIS SEQ ID TargetID # Region NO Site Sequence NO 180548 5′UTR 222 9 agatctcttggagcatgtgg223 180549 5′UTR 222 71 cttctctcgttcatttccgg 224 180550 5′UTR 222 126cttctttcatttcaggacgg 225 180551 5′UTR 222 161 tccctcctcatcctgatagg 226180552 5′UTR 222 191 cctccagacgtactccagct 227 180553 5′UTR 222 211aggaccatgagaatgatgtt 228 180554 5′UTR 222 231 acaggcctcccaagtgcagc 229180555 5′UTR 222 250 ggaaccagtatgatcccgta 230 180557 5′UTR 222 291agtagaaaatcccgaagagg 231 180558 5′UTR 222 321 cggctgtgatgcccagagcg 232180559 5′UTR 222 341 gctccagaggcgatgagccc 233 180560 5′UTR 222 361cgagccttgtaagttctgtg 234 180561 5′UTR 222 391 gcaatgattaggaagatccg 235180562 5′UTR 222 421 tacacgtcattttggaacgc 236 180563 5′UTR 222 441ggtgatctcgggcccagtcg 237 180564 5′UTR 222 471 cgtgtgtttctgagaacttg 238180565 5′UTR 222 591 cggctttcaggtcagacatg 239 180566 5′UTR 222 611ctggaacatcaccagcttct 240 180567 5′UTR 222 648 agcacatcagcaggaggccg 241180568 5′UTR 222 651 tgaagcacatcagcaggagg 242 180569 5′UTR 222 691gtctcgccccagcagtacca 243 180570 5′UTR 222 741 gcaccagagtgtatcgcaag 244180571 5′UTR 222 761 caccagccaggtggcgttga 245 180572 5′UTR 222 781tagagatgcgcggcactgtt 246 180573 5′UTR 222 812 ttgaatgttcttgtcgtagg 247180574 Start 222 855 cctcgcccacggcacccagg 248 Codon 180575 Coding 222869 gtagttgtggaagccctcgc 249 180576 Coding 222 881 gaaggtgtggtggtagttgt250 180577 Coding 222 911 gcggtactcactggcagagt 251 180578 Coding 222 929ggtgaagttgatgtgccagc 252 180579 Coding 222 1011 tcctggctaagacagtagcc 253180580 Coding 222 1031 cccgtctccagttctcttaa 254 180581 Coding 222 1039ttgtgactcccgtctccagt 255 180582 Coding 222 1049 tcagctactcttgtgactcc 256

In a further embodiment of the present invention, a series ofoligonucleotides was designed to target different regions of the mousestearoyl-CoA desaturase RNA, using published sequences (GenBankAccession number M21280.1, incorporated herein as SEQ ID NO: 216;GenBank Accession number M21281.1, incorporated herein as SEQ ID NO:217; GenBank Accession number M21282.1, incorporated herein as SEQ IDNO: 218; GenBank Accession number M21283.1, incorporated herein as SEQID NO: 219; GenBank Accession number M21284.1, incorporated herein asSEQ ID NO: 220; GenBank Accession number M21285.1, incorporated hereinas SEQ ID NO: 221; the concatenation of SEQ ID NOs 216, 217, 218, 219,220 and 221, incorporated herein as SEQ ID NO: 222). Theoligonucleotides are shown in Table 4. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 4 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Probes and primers to mouse stearoyl-CoA desaturase were designed tohybridize to a mouse stearoyl-CoA desaturase sequence, using publishedsequence information (SEQ ID NO: 222). For mouse stearoyl-CoA desaturasethe PCR primers were:

-   forward primer: ACACCAGAGACATGGGCAAGT (SEQ ID NO: 257)-   reverse primer: CATCACACACTGGCTTCAGGAA (SEQ ID NO: 258) and the PCR    probe was: FAM-CTGAAGTGAGGTCCATTAG-TAMRA (SEQ ID NO: 259) where FAM    (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent    reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.)    is the quencher dye. For mouse GAPDH the PCR primers were:-   forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:260)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:261) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID No: 262)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

The compounds were analyzed for their effect on mouse stearoyl-CoAdesaturase mRNA levels in b.END cells by quantitative real-time PCR asdescribed in other examples herein. The positive control oligonucleotideis ISIS 18078 (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 82), a 2′-O-methoxylgapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioatebackbone, which is targeted to human Jun-N-terminal kinase-2 (JNK2).Data are averages from two experiments and are shown in Table 4. Ifpresent, “N.D.” indicates “no data”. TABLE 4 Inhibition of mousestearoyl-CoA desaturase mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap Target Control IsisSEQ ID Target % SEQ ID SEQ ID # Region NO Site Sequence Inhib NO NO185154 exon: intron 216 876 ggaagctcacctcttggagc 48 263 82 185155 exon:intron 217 269 ctgctcaccgaagagggcag 0 264 82 185156 intron: exon 218 1gtagtagaaaatccctgcaa 6 265 82 185157 exon: intron 219 202tcccttacctcctctggaac 0 266 82 185158 exon: intron 220 228tgacttacccacggcaccca 41 267 82 185159 intron: exon 221 1gtggaagccctcgcctgcaa 83 268 82 185160 5′UTR 222 68 ctggctaccgccactcacaa0 269 82 185161 5′UTR 222 142 aagcctaggactttggtctg 51 270 82 1851625′UTR 222 148 gtgtgcaagcctaggacttt 0 271 82 185163 5′UTR 222 156taggaattgtgtgcaagcct 0 272 82 185164 5′UTR 222 275 atctgctgttccctctgcct0 273 82 185165 5′UTR 222 445 tccagagtagaccttggagg 15 274 82 1851665′UTR 222 571 ctagccaaggaagccaggcg 12 275 82 185167 5′UTR 222 581gcagagatagctagccaagg 2 276 82 185168 5′UTR 222 612 ttttatcggctgccagcaaa41 277 82 185169 5′UTR 222 644 ggatgaccgtgttcagtatt 41 278 82 1851705′UTR 222 697 tggctgtgcacagatctcct 82 279 82 185171 5′UTR 222 708tcagcccggtctggctgtgc 56 280 82 185172 5′UTR 222 748 gcgcttggaaacctgccctc59 281 82 185173 5′UTR 222 768 tgtaggcgagtggcggaact 43 282 82 1851745′UTR 222 795 gtggacttcggttccggagc 31 283 82 185175 5′UTR 222 830ttgctcgcctcactttccca 79 284 82 185176 5′UTR 222 854 gtgggccggcatgatgatag67 285 82 185177 5′UTR 222 877 gaactggagatctcttggag 51 286 82 185178Coding 222 1150 tagaaaatcccgaagagggc 0 287 82 185179 Coding 222 1160ggtcatgtagtagaaaatcc 10 288 82 185180 Coding 222 1165gcgctggtcatgtagtagaa 40 289 82 185181 Coding 222 1676ggattgaatgttcttgtcgt 81 290 82 185182 Coding 222 1681tcccgggattgaatgttctt 46 291 82 185183 Coding 222 1688gatattctcccgggattgaa 39 292 82 185184 Coding 222 1858gtagccttagaaactttctt 52 293 82 185185 Stop Codon 222 1918cccaaagctcagctactctt 65 294 82 185186 3′UTR 222 1934aacaggaactcagaagccca 90 295 82 185187 3′UTR 222 1967cagaatattaaatctctgcc 48 296 82 185188 3′UTR 222 1984agttgttagttaatcaacag 0 297 82 185189 3′UTR 222 2159 aattgtatatgcatttatca62 298 82 185190 3′UTR 222 2208 ctgtatagaatgttcaaatt 2 299 82 1851913′UTR 222 2236 acagcatgttccttggcttt 51 300 82 185192 3′UTR 222 2246tagcatcaaaacagcatgtt 46 301 82 185193 3′UTR 222 2259accatgctcaccctagcatc 45 302 82 185194 3′UTR 222 2408aaggatcagtatttcagaaa 39 303 82 185195 3′UTR 222 2552tctctcgagacaatctactt 67 304 82 185196 3′UTR 222 2821cttcagttaccaaaagctaa 37 305 82 185197 3′UTR 222 2887aaatgtcagctgtttagtta 0 306 82 185198 3′UTR 222 3002 ggcaacccaggcaacacctc39 307 82 185199 3′UTR 222 3017 gccacgaaagaaactggcaa 23 308 82 1852003′UTR 222 3102 atgttccccaagggcttcat 86 309 82 185201 3′UTR 222 3112tccctggcagatgttcccca 76 310 82 185202 3′UTR 222 3427ctggctctgcttcctgaagc 48 311 82 185203 3′UTR 222 3569gctgagctgttaactcacaa 71 312 82 185204 3′UTR 222 3640cacacaccgagacagatcaa 79 313 82 185205 3′UTR 222 3828caggaagcagaccctcttcc 42 314 82 185206 3′UTR 222 3958aatactgatgtgatgttttc 65 315 82 185207 3′UTR 222 3968atggttctaaaatactgatg 36 316 82 185208 3′UTR 222 4046actgagtgtttggcacctta 79 317 82 185209 3′UTR 222 4066ggctctgattctacaagtga 61 318 82 185210 3′UTR 222 4116tcaacaaaacagctcagagc 83 319 82 185211 3′UTR 222 4127gattttctacttcaacaaaa 56 320 82 185212 3′UTR 222 4333cttaaagacaccaggacctc 56 321 82 185213 3′UTR 222 4387catctggaaactgttataaa 45 322 82 185214 3′UTR 222 4466ctaagggaaggagtgagact 42 323 82 185215 3′UTR 222 4608ttacttcccaccaaatttga 59 324 82 185216 3′UTR 222 4652tgacaatgataacgaggacg 81 325 82 185217 3′UTR 222 4760cagatggtggctttgctaac 0 326 82 185218 3′UTR 222 4825 ttgttacaagagaaaggata68 327 82 185219 3′UTR 222 4884 tcagatacttagcccaggag 74 328 82 1852203′UTR 222 4902 tgttgagatgtgagactgtc 50 329 82 185221 3′UTR 222 5010cacctcagaactgcccttga 73 330 82 185222 3′UTR 222 5018gctctaatcacctcagaact 84 331 82 185223 3′UTR 222 5074ggagtctgtatgaatacctc 64 332 82 185224 3′UTR 222 5132tctctgggaagagcaatgta 58 333 82 185225 3′UTR 222 5170gtaggtagtcttgcactttg 36 334 82 185226 3′UTR 222 5211aggaagggaaaggtttcctg 38 335 82 185227 3′UTR 222 5268tacacttgggtcacaaataa 49 336 82 185228 3′UTR 222 5280aatcatccaaattacacttg 50 337 82 185229 3′UTR 222 5303cttcaagagttgatattaat 60 338 82 185230 3′UTR 222 5329atacaatctcaatcagtaca 76 339 82 185231 3′UTR 222 5347cacttttattaggaacaaat 0 340 82

As shown in Table 4, SEQ ID NOs 263, 267, 268, 270, 277, 278, 279, 280,281, 282, 284, 285, 286, 289, 290, 291, 293, 294, 295, 296, 298, 300,301, 302, 304, 309, 310, 311, 312, 313, 314, 315, 317, 318, 319, 320,321, 322, 323, 324, 325, 327, 328, 329, 330, 331, 332, 333, 336, 337,338, 339 demonstrated at least 40% inhibition of stearoyl-CoA desaturasein this experiment and are therefore preferred. More preferred are SEQID NOs 295, 331 and 268. The target regions to which these preferredsequences are complementary are herein referred to as “preferred targetsegments” and are therefore preferred for targeting by compounds of thepresent invention.

In a further embodiment of the present invention, a series ofoligonucleotides was designed to target different regions of the mousestearoyl-CoA desaturase RNA, using published sequences (GenBankAccession number M21280.1, incorporated herein as SEQ ID NO: 216;GenBank Accession number M21281.1, incorporated herein as SEQ ID NO:217; GenBank Accession number M21282.1, incorporated herein as SEQ IDNO: 218; GenBank Accession number M21283.1, incorporated herein as SEQID NO: 219; GenBank Accession number M21284.1, incorporated herein asSEQ ID NO: 220; GenBank Accession number M21285.1, incorporated hereinas SEQ ID NO: 221; the concatenation of SEQ ID NOs 216, 217, 218, 219,220 and 221, incorporated herein as SEQ ID NO: 222). Theoligonucleotides are shown in Table 5. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 5 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Probes and primers to mouse stearoyl-CoA desaturase were designed tohybridize to a mouse stearoyl-CoA desaturase sequence, using publishedsequence information (SEQ ID NO: 222). For mouse stearoyl-CoA desaturasethe PCR primers were:

-   forward primer: TTCCGCCACTCGCCTACA (SEQ ID NO: 341)-   reverse primer: CTTTCCCAGTGCTGAGATCGA (SEQ ID NO: 342) and the PCR    probe was: FAM-CAACGGGCTCCGGAACCGAA-TAMRA (SEQ ID NO: 343) where FAM    (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent    reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.)    is the quencher dye. For mouse GAPDH the PCR primers were:-   forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:261)-   reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:262) and the PCR    probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 263)    where JOE is the fluorescent reporter dye and TAMRA is the quencher    dye.

The compounds were analyzed for their effect on mouse stearoyl-CoAdesaturase mRNA levels in primary mouse hepatocytes by quantitativereal-time PCR as described in other examples herein. The positivecontrol oligonucleotide is ISIS 18078 (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:82), a 2′-O-methoxyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone, which is targeted to human Jun-N-terminalkinase-2 (JNK2). Data are averages from two experiments and are shown inTable 5. If present, “N.D.” indicates “no data”. TABLE 5 Inhibition ofmouse stearoyl-CoA desaturase mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap Target Control IsisSEQ ID Target % SEQ SEQ ID # Region NO Site Sequence Inhib ID NO NO180556 5′UTR 222 261 gcttgcaggagggaaccagt 40 344 82 244459 5′UTR 222 138ctaggactttggtctggcgc 6 345 82 244461 5′UTR 222 280 gcgcaatctgctgttccctc0 346 82 244464 5′UTR 222 401 agggcgcgctgctccaaccc 0 347 82 244467 5′UTR222 462 aagaaagccaagtagattcc 0 348 82 244470 5′UTR 222 692gtgcacagatctcctgggct 35 349 82 244472 5′UTR 222 736 ctgccctcctgactctcggg54 350 82 244476 Coding 222 878 agaactggagatctcttgga 71 351 82 244479Coding 222 1020 cctcctcatcctgataggtg 16 352 82 244481 Coding 222 1045acgtactccagcttgggcgg 50 353 82 244484 Coding 222 1057atgttcctccagacgtactc 33 354 82 244487 Coding 222 1062gaatgatgttcctccagacg 43 355 82 244490 Coding 222 1068ccatgagaatgatgttcctc 50 356 82 244493 Coding 222 1098tcccgtacaggcctcccaag 54 357 82 244495 Coding 222 1128tgtagagcttgcaggaggga 10 358 82 244498 Coding 222 1264gccatggtgttggcaatgat 41 359 82 244501 Coding 222 1324tctgagaacttgtggtgggc 18 360 82 244504 Coding 222 1329gtgtttctgagaacttgtgg 50 361 82 244507 Coding 222 1334ggcgtgtgtttctgagaact 63 362 82 244510 Coding 222 1347aattgtgagggtcggcgtgt 11 363 82 244514 Coding 222 1357ccacggcgggaattgtgagg 47 364 82 244517 Coding 222 1363aagaagccacggcgggaatt 16 365 82 244520 Coding 222 1387agcagccaacccacgtgaga 51 366 82 244523 Coding 222 1395tgcgcacaagcagccaaccc 67 367 82 244526 Coding 222 1400gtgtttgcgcacaagcagcc 52 368 82 244528 Coding 222 1408acagccgggtgtttgcgcac 63 369 82 244532 Coding 222 1413ctttgacagccgggtgtttg 3 370 82 244535 Coding 222 1418cttctctttgacagccgggt 50 371 82 244538 Coding 222 1423ccgcccttctctttgacagc 64 372 82 244541 Coding 222 1435atgtccagttttccgccctt 55 373 82 244542 Coding 222 1440cagacatgtccagttttccg 55 374 82 244546 Coding 222 1445caggtcagacatgtccagtt 48 375 82 244549 Coding 222 1450gctttcaggtcagacatgtc 49 376 82 244553 Coding 222 1455tctcggctttcaggtcagac 50 377 82 244554 Coding 222 1460cagcttctcggctttcaggt 37 378 82 244557 Coding 222 1465atcaccagcttctcggcttt 20 379 82 244560 Coding 222 1470ggaacatcaccagcttctcg 42 380 82 244565 Coding 222 1477ctcctctggaacatcaccag 17 381 82 244567 Coding 222 1486ttgtagtacctcctctggaa 0 382 82 244569 Coding 222 1516aggatgaagcacatcagcag 29 383 82 244572 Coding 222 1525agcgtgggcaggatgaagca 45 384 82 244577 Coding 222 1538gtaccagggcaccagcgtgg 37 385 82 244578 Coding 222 1543cagcagtaccagggcaccag 25 386 82 244581 Coding 222 1548cgccccagcagtaccagggc 13 387 82 244585 Coding 222 1583gaaggtgctaacgaacaggc 18 388 82 244589 Coding 222 1627ctgttcaccagccaggtggc 37 389 82 244591 Coding 222 1633gcggcactgttcaccagcca 3 390 82 244595 Coding 222 1693accaggatattctcccggga 62 391 82 244598 Coding 222 1732tggtagttgtggaagccctc 54 392 82 244599 Coding 222 1768tactcactggcagagtagtc 19 393 82 244602 Coding 222 1773agcggtactcactggcagag 31 394 82 244607 Coding 222 1778gtgccagcggtactcactgg 5 395 82 244609 Coding 222 1783ttgatgtgccagcggtactc 29 396 82 244613 Coding 222 1792gtggtgaagttgatgtgcca 40 397 82 244615 Coding 222 1798aagaacgtggtgaagttgat 12 398 82 244619 Coding 222 1860cagtagccttagaaactttc 75 399 82 244620 Coding 222 1885ccagttctcttaatcctggc 63 400 82 244623 Coding 222 1891ccgtctccagttctcttaat 37 401 82 244626 3′UTR 222 2006taacaccccgatagcaatat 59 402 82 244630 3′UTR 222 2365gagggtggacagacacaggc 4 403 82 244633 3′UTR 222 2445 cttgaagctaggaacaagga68 404 82 244636 3′UTR 222 2647 tatggctacctctctctctc 82 405 82 2446393′UTR 222 2920 ttttcatagtttcacaccat 47 406 82 244643 3′UTR 222 2970tattttctaagtgaaatagt 5 407 82 244644 3′UTR 222 3243 taggcagcactaggcaggct33 408 82 244647 3′UTR 222 3373 aggaacaggcctggacagca 36 409 82 2446503′UTR 222 4168 gagggctataggtcagtaga 34 410 82 244655 3′UTR 222 4329aagacaccaggacctcaatg 18 411 82 244656 3′UTR 222 4532ccaatgtactgatgactctc 62 412 82 244660 3′UTR 222 4737tcacaccacctcactggagc 62 413 82 244663 3′UTR 222 4987agtaggtcagtattaataac 35 414 82 244667 3′UTR 222 5220atctcattcaggaagggaaa 0 415 82 244668 3′UTR 222 5272 aaattacacttgggtcacaa57 416 82 244673 3′UTR 222 5326 caatctcaatcagtacaagt 37 417 82

As shown in Table 5, SEQ ID NOs 344, 350, 351, 353, 355, 356, 357, 359,361, 362, 364, 366, 367, 368, 369, 371, 372, 373, 374, 375, 376, 377,380, 384, 391, 392, 397, 399, 400, 402, 404, 405, 406, 412, 413, 416exhibited at least 40% inhibition of stearoyl-CoA desaturase in thisexperiment. A more preferred sequence is SEQ ID NO: 373. The targetregions to which these preferred sequences are complementary are hereinreferred to as “preferred target segments” are therefore preferred fortargeting by compounds of the present invention.

Example 19 Effects of Antisense Inhibition of Mouse Stearoyl-CoADesaturase Expression in Mice: mRNA Levels in Liver and Fat Tissue

Ob/ob mice harbor a mutation in the leptin gene. The leptin mutation ona C57B/16 background yields a db/db phenotype, characterized by,hyperglycemia, obesity, hyperlipidemia, and insulin resistance. However,a mutation in the leptin gene on a different mouse background canproduce obesity without diabetes, and these mice are referred to asob/ob mice. Leptin is a hormone that regulates appetite. Leptindeficiency results in obesity in animals and humans.

In accordance with the present invention, ISIS 185222 (SEQ ID NO: 332)was further investigated for its ability to reduce target levels inliver and fat tissue in ob/ob mice maintained on a high-fat (11% kcalfrom fat) or low-fat (2% from fat) diet.

ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 418) is a scrambledcontrol oligonucleotide. ISIS 141923 is a chimeric oligonucleotide(“gapmer”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

Eight-week old male ob/ob mice were dosed twice weekly byintraperitoneal injection with saline or 25 mg/kg of ISIS 185222 or ISIS141923. Mice were maintained on a low-fat or high-fat diet. At the endof the ten-week investigation period, mice were sacrificed and evaluatedfor stearoyl-CoA desaturase and stearoyl-CoA desaturase-2 mRNA levels inliver and fat tissue. Inhibition of mRNA expression was determined byquantitative real-time PCR as described in other examples herein. Thedata are the averages of mRNA levels from nine mice per group and arepresented in Table 6. TABLE 6 Antisense inhibition of stearoyl-CoAdesaturase in mouse liver and fat tissue Percent Inhibition Saline ISISISIS control 185222 141923 mRNA Diet Liver Fat Liver Fat Liver Fatstearoyl-CoA High-fat 0 0 93 96 29 28 desaturase Low-fat 0 0 94 98 0 0stearoyl-CoA High-fat 0 0 37 40 52 5 desaturase-2 Low-fat 0 0 37 0 0 0

The data demonstrate that the oligonucleotide of the present inventioncan inhibit the expression of stearoyl-CoA desaturase in vivo, in bothliver and fat tissues. The data also suggest that antisense inhibitionof stearoyl-CoA desaturase can reduce expression of stearoyl-CoAdesaturase-2.

Example 20 Effects of Antisense Inhibition of Stearoyl-CoA Desaturase ina Mouse Model of Obesity: Organ Weights and Levels of Serum Cholesterol,Triglyceride and Liver Enzymes

In accordance with the present invention, further investigation of theeffects antisense inhibition of stearoyl-CoA desaturase was conducted inob/ob mice. The saline-treated and antisense oligonucleotide-treatedob/ob mice described in Example 19 were also evaluated for body organweight, levels of serum cholesterol and triglyceride and levels of liverenzymes ALT and AST at the end of the ten-week investigation period.Increased levels of ALT and AST are indicative of impaired liverfunction. Blood samples were collected and evaluated for cholesterol,triglyceride, ALT and AST levels. White adipose tissue (WAT), spleen andliver were individually weighed. Data are expressed as percent changerelative to the saline control for the respective diet. The datarepresent the average of nine mice per treatment group and are presentedin Table 7. TABLE 7 Effects of antisense inhibtion of stearoyl-CoAdesaturase on cholesterol, triglyceride, ALT, AST and organ weightPercent Change Liver Organ Enzymes Weight ALT AST CHOL TRIG Liver SpleenWAT ISIS High-fat −76 −72 −11 3 −8 19 2 185222 Low-fat −60 −55 15 29 −29−19 −4 ISIS High-fat −42 −39 −1 −10 −4 20 9 141923 Low-fat 45 34 50 7031 −41 27

The data demonstrate that, concomitant with reducing target mRNAexpression (shown in Example 19), the oligonucleotide of the presentinvention lowers the levels of the liver enzyme ALT and AST in animalsmaintained on either a high-fat or low-fat diet, which is indicative ofimproved liver function. Histologically, mice treated with ISIS 185222and maintained on a low-fat diet exhibit lowered hepatic fattydegeneration.

Example 21 Effects of Antisense Inhibition of Stearoyl-CoA Desaturase ina Mouse Model of Obesity: Plasma Glucose and Insulin, Body Weight, FoodConsumption and Oxygen Consumption

In accordance with the present invention, the ob/ob mice described inExample 19 were further evaluated to assess the effects of antisenseinhibition of stearoyl-CoA desaturase.

Mice were evaluated for plasma glucose and oxygen consumption followingthree weeks of treatment. The glucose evaluation was conducted followingan overnight fast. The oxygen consumption was determined by measuringmetabolic rate (MR) and respiratory quotient (RER) during both light anddark cycles. Plasma glucose and insulin (both in non-fasting mice), foodconsumption, oxygen consumption and total body weight were measuredthroughout the ten-week treatment period. Shown in Table 8 are plasmainsulin (non-fasting) following five weeks of treatment, foodconsumption following six weeks of treatment and plasma glucose(non-fasting) and total body weight following seven weeks of treatment.The data are the averages of measurements from seven to nine mice andare expressed as percent change relative to saline control for therespective diet. The data are presented in Table 8. TABLE 8 Effects ofantisense inhibtion of stearoyl-CoA desaturase on body weight, foodconsumption, insulin, glucose and oxygen consumption Percent ChangeWeight Oxygen consumption Total Food Insulin Glucose MR RER BodyConsumed Fed Fed Fast Dark Light Dark Light ISIS High-fat −3 −10 −3 2 −33 0 0 2 185222 Low-fat −60 −55 15 29 −29 4 11 −6 0 ISIS High-fat −42 −39−1 −10 −4 −5 −5 3 0 141923 Low-fat 45 34 50 70 31 6 5 2 0

The data suggest that body weight and food consumption are lowered bytreatment of ob/ob mice with the oligonucleotide of the presentinvention. Comparison of blood glucose, insulin and oxygen consumptionin mice fed the same diet does not reveal any significant changesbetween saline-treated and antisense oligonucleotide-treated mice.

Example 22 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group, which has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine, which notonly cleaves the oligonucleotide from the solid support but also removesthe acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 23 Design and Screening of Duplexed Antisense CompoundsTargeting Stearoyl-CoA Desaturase

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target stearoyl-CoA desaturase. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 1. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand    |||||||||||||||||||TTgctctccgcctgccctggc Complement

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of the buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate stearoyl-CoA desaturase expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 .L of OPTI-MEM-1 medium containing 12μg/mL LIPOFECTIN reagent (Gibco BRL) and the desired duplex antisensecompound at a final concentration of 200 nM. After 5 hours of treatment,the medium is replaced with fresh medium. Cells are harvested 16 hoursafter treatment, at which time RNA is isolated and target reductionmeasured by RT-PCR.

Example 24 Design of Phenotypic Assays and in vivo Studies for the useof Stearoyl-CoA Desaturase Inhibitors Phenotypic Assays

Once stearoyl-CoA desaturase inhibitors have been identified by themethods disclosed herein, the compounds are further investigated in oneor more phenotypic assays, each having measurable endpoints predictiveof efficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of stearoyl-CoA desaturase in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withstearoyl-CoA desaturase inhibitors identified from the in vitro studiesas well as control compounds at optimal concentrations which aredetermined by the methods described above. At the end of the treatmentperiod, treated and untreated cells are analyzed by one or more methodsspecific for the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status, which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the stearoyl-CoA desaturaseinhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

In vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor stearoyl-CoA desaturase inhibitor. Furthermore, to prevent thedoctors from being biased in treatments, they are not informed as towhether the medication they are administering is a stearoyl-CoAdesaturase inhibitor or a placebo. Using this randomization approach,each volunteer has the same chance of being given either the newtreatment or the placebo.

Volunteers receive either the stearoyl-CoA desaturase inhibitor orplacebo for eight week period with biological parameters associated withthe indicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingstearoyl-CoA desaturase or stearoyl-CoA desaturase protein levels inbody fluids, tissues or organs compared to pre-treatment levels. Othermeasurements include, but are not limited to, indices of the diseasestate or condition being treated, body weight, blood pressure, serumtiters of pharmacologic indicators of disease or toxicity as well asADME (absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and stearoyl-CoA desaturase inhibitor treatment.In general, the volunteers treated with placebo have little or noresponse to treatment, whereas the volunteers treated with thestearoyl-CoA desaturase inhibitor show positive trends in their diseasestate or condition index at the conclusion of the study.

1. A compound 8 to 50 nucleobases in length targeted to a nucleic acidmolecule encoding human stearoyl-CoA desaturase, wherein the compoundspecifically hybridizes with a nucleic acid molecule encoding humanstearoyl-CoA desaturase and inhibits the expression of humanstearoyl-CoA desaturase.
 2. The compound according to claim 1, which isan antisense oligonucleotide.
 3. The compound according to claim 2,which hybridizes to a sequence within a nucleic acid molecule encodinghuman stearoyl-CoA desaturase SEQ ID NO: 3, provided that said sequencedoes not include nucleotide sequences spanning 70 through nucleotide 91,nucleotide 242 through nucleotide 262, or nucleotide 860 throughnucleotide 882 of SEQ ID NO:
 3. 4. The compound according to claim 2,wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The compound according to claim 4, whereinthe modified internucleoside linkage is a phosphorothioate linkage. 6.The compound according to claim 2, wherein the antisense oligonucleotidecomprises at least one modified sugar moiety.
 7. The compound accordingto claim 6, wherein the modified sugar moiety is a 2′-O-methoxyethylsugar moiety.
 8. The compound according to claim 2, wherein theantisense oligonucleotide comprises at least one modified nucleobase. 9.The compound according to claim 8, wherein the modified nucleobase is a5-methylcytosine.
 10. The compound according to claim 2, wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 11. Thecompound according to claim 2, wherein said antisense oligonucleotideinhibits expression of said human stearoyl CoA desaturase by at least10% in a suitable assay.
 12. The compound according to claim 2, whereinsaid antisense oligonucleotide inhibits expression of said humanstearoyl CoA desaturase by at least 90% in a suitable assay.
 13. Thecompound according to claim 1, wherein said compound comprises asequence selected from the group consisting of SEQ ID NOS: 18, 19, 20,23, 25, 26, 29, 30, 31, 33, 39, 43, 44, 83, 84, 85, 87, 88, 89, 91, 93,94, 95, 97, 98, 100, 101, 102, 103, 105, 107, 108, 109, 110, 112, 113,114, 115, 117, 118, 119, 120, 124, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 149, 150, 153, 154, 157,158, 159, 160, 164, 165, 167, 168, 169, 188, 189, 197, 204, 207 and 210.14. The compound according to claim 1, wherein said compound comprisesan antisense nucleic acid molecule that is specifically hybridizablewith a 5′-untranslated region (5′ UTR) of stearoyl CoA desaturase. 15.The compound according to claim 1, wherein said compound comprises anantisense nucleic acid molecule that is specifically hybridizable with astart codon region of stearoyl CoA desaturase.
 16. The compoundaccording to claim 1, wherein said compound comprises an antisensenucleic acid molecule that is specifically hybridizable with a codingregion of stearoyl CoA desaturase.
 17. The compound according to claim1, wherein said compound comprises an antisense nucleic acid moleculethat is specifically hybridizable with a stop codon region of stearoylCoA desaturase.
 18. The compound according to claim 1, wherein saidcompound comprises an antisense nucleic acid molecule that isspecifically hybridizable with a 3′-untranslated region (3′ UTR) ofstearoyl CoA desaturase.
 19. A compound 8 to 50 nucleobases in lengthwhich specifically hybridizes with at least an 8-nucleobase portion ofan active site on a nucleic acid molecule encoding human stearoyl-CoAdesaturase.
 20. The compound according to claim 19, wherein said portionof said active site falls outside of nucleotide sequences spanning 70through nucleotide 91, nucleotide 242 through nucleotide 262, ornucleotide 860 through nucleotide 882 of a nucleic acid moleculeencoding human stearoyl-CoA desaturase SEQ ID NO:
 3. 21. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 22. The composition according to claim 21, furthercomprising a colloidal dispersion system.
 23. The composition accordingto claim 12, wherein the compound is an antisense oligonucleotide.
 24. Amethod of inhibiting the expression of human stearoyl-CoA desaturase incells or tissues comprising contacting the cells or tissues with thecompound of claim 1 so that expression of human stearoyl-CoA desaturaseis inhibited.
 25. A method of treating a human having a disease orcondition associated with human stearoyl-CoA desaturase comprisingadministering to the human a therapeutically or prophylacticallyeffective amount of the compound of claim 1 so that expression of humanstearoyl-CoA desaturase is inhibited.
 26. The method according to claim25, wherein the condition involves abnormal lipid metabolism.
 27. Themethod according to claim 25, wherein the condition involves abnormalcholesterol metabolism.
 28. The method according to claim 25, whereinthe condition is atherosclerosis.
 29. The method according to claim 25,wherein the disease is cardiovascular disease.
 30. A method of screeningfor an antisense compound, the method comprising the steps of: a.contacting a preferred target region of a nucleic acid molecule encodinghuman stearoyl-CoA desaturase with one or more candidate antisensecompounds, said candidate antisense compounds comprising at least an8-nucleobase portion which is complementary to said preferred targetregion, and b. selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encoding humanstearoyl-CoA desaturase.
 31. A method for improving liver function in ananimal having elevated liver enzymes comprising administering to saidanimal a therapeutically or prophylactically effective amount of thecompound of claim 1 so that expression of stearoyl CoA desaturase isinhibited and thereby lowers liver enzyme levels.
 32. A method fortreating an obese animal comprising administering to said animal atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of stearoyl CoA desaturase is inhibited,thereby reducing said animal's weight and appetite.
 33. A duplexedantisense compound comprising: (a) a nucleobase sequence 8 to 80nucleobases in length targeted to a nucleic acid molecule encodingstearoyl CoA desaturase with at least one natural or modified nucleobaseforming an overhang at a terminus of said sequence; and (b) thecomplementary sequence of said sequence (a) having optionally at leastone natural or modified nucleobase forming an overhang at a terminus ofsaid complementary sequence; wherein said sequences (a) and (b), whenhybridized, have at least one single-stranded overhang at at least oneof terminus of said hybridized duplex, and wherein said duplex wheninteracted with a nucleic acid molecule encoding stearoyl CoA desaturasecan modulate the expression of said reductase.